Activation of Phospholipase D in Rat Thymocytes by Sphingosine

Lee, Young-kyun,Choi, Myung-Un Korean Chemical Society 2002 Bulletin of the Korean Chemical Society Vol.23 No.10

Sphingosine is known to regulate a wide range of cell physiology including growth, differentiation, and apoptosis. In this study, we examined the effect of sphingosine on the phospholipase D (PLD) activity in rat thymocytes. Sphingosine potently stimulated PLD in the absence of extracellular calcium, while depletion of intracellular calcium by BAPTA/AM treatment completely blocked activation of PLD by sphingosine. Sphingosine-induced increase of the intracellular calcium concentration was confirmed using a fluorescent calcium indicator Fluo-3/AM. A phosphoinositide-specific phospholipase C inhibitor U73122 partially inhibited the stimulation of PLD by sphingosine. When mouse PLD2 gene was transfected into mouse thymoma EL4 cells, which lack intrinsic PLD activity, sphingosine could stimulate PLD2 significantly while overexpression of human PLD1 had no effect. Taken together, the sphingosine-stimulated PLD activity in rat thymocytes is dependent on the mobilization of intracellular calcium and appears to be due to the PLD2 isoform.

Effects of Sphingosine-1-phosphate on Vestibular Nuclear Neurons

Jae-Hyuk Lee,Sujeong Jang,Song-Hee Kim,Han-Seong Jeong,Jong-Seong Park(박종성) 대한의생명과학회 2010 Biomedical Science Letters Vol.16 No.1

This study was designed to investigate the effects of sphingosine-1-phosphate on the neuronal activity of rat medial vestibular nuclear neurons. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated medial vestibular nuclear neurons were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes respectively. 15 medial vestibular nuclear neurons revealed excitatory responses to 1 and 5 μM of sphingosine-1-phosphate. The spike frequency and resting membrane potential of these cells were increased by sphingosine-1-phosphate. The amplitude of afterhyperpolarization was decreased by sphingosine-1-phosphate. Whole potassium currents of medial vestibular nuclear neurons were decreased by sphingosine-1-phosphate (n=12). Sphingosine-1-phosphate did not affect the charybdotoxin-treated potassium currents. These experimental results suggest that sphingosine-1-phosphate increases the neuronal activity of the medial vestibular nuclear neurons by altering the resting membrane potential and afterhyperpolarization.

Sphingosine Kinase: Biochemical and Cellular Regulation and Role in Disease

Taha, Tarek Assad,Hannun, Yusuf Awni,Obeid, Lina Marie Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.2

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.

Sphingosine-1-phosphate의 HaCaT 세포의 증식에 대한 이중적 효과에 대한 연구

김형훈,강숙경,장성은,이미우,최지호,성경제,문기찬,고재경 大韓皮膚硏究學會 2003 大韓皮膚硏究學會誌 Vol.10 No.4

Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation

홍석우,이진미,권혜미,박세은,이은정,박철영,오기원,박성우,이원영 대한내분비학회 2018 Endocrinology and metabolism Vol.33 No.3

Background: Emerging evidence suggests that sphingolipids may be involved in type 2 diabetes. However, the exact signaling defectthrough which disordered sphingolipid metabolism induces β-cell dysfunction remains unknown. The current study demonstratedthat sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SphK), is an essential factor for maintaining β-cell functionand survival via regulation of mitochondrial action, as mediated by prohibitin (PHB). Methods: We examined β-cell function and viability, as measured by mitochondrial function, in mouse insulinoma 6 (MIN6) cellsin response to manipulation of cellular S1P and PHB levels. Results: Lack of S1P induced by sphingosine kinase inhibitor (SphKi) treatment caused β-cell dysfunction and apoptosis, with repressionof mitochondrial function shown by decreases in cellular adenosine triphosphate content, the oxygen consumption rate, theexpression of oxidative phosphorylation complexes, the mitochondrial membrane potential, and the expression of key regulators ofmitochondrial dynamics (mitochondrial dynamin-like GTPase [OPA1] and mitofusin 1 [MFN1]). Supplementation of S1P led to therecovery of mitochondrial function and greatly improved β-cell function and viability. Knockdown of SphK2 using small interferingRNA induced mitochondrial dysfunction, decreased glucose-stimulated insulin secretion (GSIS), and reduced the expression ofPHB, an essential regulator of mitochondrial metabolism. PHB deficiency significantly reduced GSIS and induced mitochondrialdysfunction, and co-treatment with S1P did not reverse these trends. Conclusion: Altogether, these data suggest that S1P is an essential factor in the maintenance of β-cell function and survival throughits regulation of mitochondrial action and PHB expression.

초고성능액체크로마토그래피-텐덤질량분석법에 의한 혈청 중스핑고신과 스핑고신-1-포스페이트의 동시 분석

나승훈(Seonghoon Na),윤혜란(Hye-Ran Yoon) 대한약학회 2021 약학회지 Vol.65 No.1

Sphingosine (SPH) and sphingosine-1-phosphate (S1P) are emerging as key players in asthma metabolism and in numerous cellular inflammation processes. To identify potential biomarkers of asthma and inflammatory therapeutics, it is essential to determine their levels. Herein, we developed a rapid and sensitive UHPLC-MS/MS method to simultaneously quantify SPH and S1P in human serum using C17-SPH and C17-S1P as internal standards. After methanol precipitation of serum proteins, the supernatants were analyzed by MS/MS performed in the positive ion mode by multiple reaction monitoring. UHPLC analysis (C18 column) was performed using two mobile phase systems (water containing 0.1% formic acid, and 85% acetonitrile containing 0.1% formic acid) within 5 min of the short run. The calibration curves were linear in the range of 0.002-1.5 µg/mL for S1P and SPH with an R 2 greater than 0.9999. The LOD and LOQ were 0.0002 and 0.0004 µg/mL for S1P, and 0.0005 and 0.001 µg/mL for SPH, respectively. The accuracy and precision of the method were in the range of 89.8-100.7% (RSD, 1.5-2.8%) for both SPH and S1P species. We were able to quantify both molecules in serum from healthy and asthmatic patients. These results suggest that SPH and S1P are promising potential biomarkers, and also contribute to the basic data for the construction of an omics-based platform for preventive index prior to asthma diagnosis.

The role of sphingosine-metabolizing enzymes in viral immunopathogenesis

Young-Jin Seo 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Diverse viruses continue to pose a threat to public health by causing illness and mortality in humans. Discovering host factors that regulate viral propagation and pathogenesis is vital for the development of novel drugs. In this study, we investigated the role of sphingosine-metabolizing enzymes in acute and chronic viral infections. We demonstrated that the inhibition of sphingosine kinase (SphK) 1 or SphK2 enhances the host defense against acute and chronic viruses. The temporal inhibition of SphK1 or SphK2 protected mice from influenza virus-induced morbidity and mortality. Furthermore, we found that SphK2 functions during chronic viral (Clone 13 strain of lymphocytic choriomeningitis virus) infection to limit T cell immune pathology, which subsequently aids in the establishment of virusinduced immunosuppression and the resultant viral persistence. Notably, oral instillation of an SphK2- selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Clone 13 persistence in mice. Collectively, our results indicate that sphingosine-metabolizing enzymes function as pro-viral factors during viral infection. Therefore, SphK1 and SphK2 represent potential host targets for therapeutics against acute and chronic viral infections.

Sphingosine-1-Phosphate-Induced Migration and Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

Hae Young Song(송해영),Sang Hun Shin(신상훈),Min Young Kim(김민영),Jae Ho Kim(김재호) 한국생명과학회 2011 생명과학회지 Vol.21 No.2

중간엽 줄기세포의 이동과 분화는 손상된 조직의 재생을 위해 필수적이다. Sphingosine-1-phosphate (S1P)는 세포성장, 생존, 분화, 이동성 등 여러 가지 생명현상에 중요한 역할을 하는 생리활성 지질이다. 본 연구에서는 인체 골수유래 중간엽 줄기세포의 이동과 세포분화에 대한 S1P의 영향을 조사하였다. S1P는 중간엽 줄기세포의 이동을 증가시켰으며 pertussis toxin의 전처리는 S1P에 의한 세포이동을 억제하였다. 본 결과는 S1P에 의한 세포이동과정에 Gi에 연결된 수용체가 관여함을 제시한다. S1P₁과 S1P₃ 수용체에 대한 길항제인 VPC23019의 전처리나 siRNA를 이용한 S1P₁ 수용체의 발현억제는 S1P에 의한 세포 내 칼슘 증가와 중간엽 줄기세포의 이동을 저해하였다. 또한, S1P의 처리는 중간엽 줄기세포에서 평활근세포의 표지유전자인 α-smooth muscle actin (α-SMA)의 발현을 증가시켰으며 VPC23019의 전처리는 S1P에 의한 α-SMA의 발현증가를 저해하였다. S1P는 중간엽 줄기세포에서 p38 mitogen-activated protein kinase (p38 MAPK)의 인산화를 촉진하였으며 p38 MAPK의 저해제인 SB202190의 전처리 또는 p38 MAPK의 dominant negative mutant의 과발현은 S1P에 의한 중간엽 줄기세포의 이동 α-SMA 발현증가를 억제하였다. 본 연구결과는 S1P가 S1P1-p38 MAPK 신호전달기전을 통해 중간엽 줄기세포의 이동과 평활근세포로의 분화를 촉진함으로써 중간엽 줄기세포를 이용한 조직재생에의 활용 가능성을 제시한다. Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]i) and pretreatment with VPC23019, an antagonist of S1P₁/S1P₃, blocked S1P-induced migration and increase of [Ca<SUP>2+</SUP>]i. Small interfering RNA-mediated knockdown of endogenous S1P1 attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced α-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and α-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an S1P1-p38 MAPK-dependent mechanism.

A Sphingosine Kinase-1 Inhibitor, SKI-II, Induces Growth Inhibition and Apoptosis in Human Gastric Cancer Cells

Li, Pei-Hua,Wu, Jin-Xia,Zheng, Jun-Nian,Pei, Dong-Sheng Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.23

SKI-II has been reported as an inhibitor of sphingosine kinase 1 and has been extensively used to prove the involvement of sphingosine kinase and sphingosine-1-phosphate (Sphk1) in cellular processes. In the current study, we investigated the effects of SKI-II and its potential mechanisms in human gastric cancer SGC7901 cells. After treatment with SKI-II, cell growth, cell cycle distribution, apoptosis, expression of Sphk1, NF-${\kappa}B$, Bcl-2, Bax and p27 were assessed by MTT assay, flow cytometry, electron microscopy, immunocytochemistry and Western-blot assay, respectively. Our results showed that SKI-II markedly inhibited SGC7901 cell survival in a dose-dependent manner, reduced cell proliferation with accumulation of cells in the G0/G1 phase and induced apoptosis in the tumor cells. Furthermore, Western blotting and immunocytochemistry showed that the expression of p27 and Bax was increased significantly, but the expression of NF-${\kappa}B$, Bcl-2 and Sphk1 decreased by different degrees. These results indicate that SKI-II induced cell growth arrest and apoptosis. The increased apoptotic sensitivity of SGC7901 was correlated with NF-${\kappa}B$ or Bcl-2/Bax activation.

Synthesis and Characterization of an Amino-oxy-modified Sphingosine-1-Phosphate Derivative that Can Replace Thiolated-S1P in Competitive ELSIA

박지혜,Yeji Lee,Eunjin Kim,Yong-Tae Kim,홍인석 대한화학회 2020 Bulletin of the Korean Chemical Society Vol.41 No.4

Sphingosine-1-phosphate (S1P) is a signaling molecule that has various roles in vivo. Recently, S1P has emerged as a biomarker in the early diagnosis of osteoporosis and cancer. A competitive enzyme-linked immunosorbent assay (ELISA) assay kit for detecting S1P in blood or in various samples is now commercially available. Competitive ELISA requires an anti-S1P antibody and S1P-coated microtiter plates. The antigenic derivative used here is the thiolated-S1P in which a sulfur hydryl group is introduced at the terminal end of the S1P hydrophobic chain. However, the sulfur hydryl group is easily oxidized, and its solubility in water is considerably low, making it difficult to handle. In this study, amino-oxy-modified S1P derivatives that can replace thiolated-S1P were synthesized. Amino-oxy groups are easy to attach to proteins and other polymers without any linker, and are easy to handle because they are not easily oxidized, and soluble in water. The synthesized S1P derivative was directly immobilized on a microtiter plate, and an ELISA assay was performed which obtained comparable results with the conventional thiolated-S1P reference. It is expected that the anti-S1P antibody can be easily induced by attaching this amino-oxy-modified S1P to reduced carrier proteins.